Mus

Control

Fig. 3. NO-induced modification of MT thiols in HL-60 cells. (A) Typical column elution profiles of reduced thiols from cell homogenates obtained from HL-60 cells under control conditions (solid stars) and after MT induction by 48-hr exposure to 150 fiM ZnCl2 (solid circles). Note the prominent appearance of an intermediate peak of reduced SH groups between high molecular weight (HMW) proteins and glutathione (GSH) in zinc-induced cells. In addition, it appears the treatment of zinc-induced cells with 1 mM NOC-15 for 1 hr (open circles) produced a substantial reduction in the SH group content of the MT peak. To specifically identify the middle peak as MT, the various column fractions were analyzed for MT immunoreactivity. (B) Only the fractions corresponding to the middle peak obtained from zinc-induced cells contain significant MT-immunoreactive protein. Furthermore, in contrast to the effect of thiol content, exposure of zinc-induced cells to NO did not alter the amount of immunoreactive MT contained within the middle peak.

represent hypersensitive thiols that may be preferentially modified during oxida-tive/nitrosative challenge. No change in immunoreactive protein is observed after NO treatment (Fig. 3B), indicating that the loss of SH groups, indeed, reflects the degree of cysteine modification within MT. Further details regarding domain specificity for these effects could be obtained by proteolytic mapping of modified MT.

Although the approaches outlined above facilitate identification of modifications of MT from intact cells, it is oftentimes desirable to obtain such information from live cells in a more dynamic fashion. To this end, we followed the example of the Ca2+ indicator, caméléon-1,25 and constructed a chimera in which a yellow-green fluorescent protein (GFP) variant, that is, enhanced yellow fluorescent protein (EYFP), and a cyan GFP variant (ECFP) were fused to the C and N termini, respectively, of human MTIIa (see Fig. 4). We used liposome-mediated transfer of this expression vector into primary cultures of sheep pulmonary artery endothelial cells and imaged these cells on a Nikon (Garden City, NY) inverted microscope with a Photometries (Roper Scientific, Trenton, NJ) cooled charge-coupled device camera controlled by ISEE software (Inovision, Raleigh, NC).26 The cyan and yellow GFPs acted as donor and acceptor, respectively, for fluorescence resonance energy transfer (FRET) and, hence, revealed changes in intramolecular proximity and relative orientation of the fluorophores. By constantly monitoring the emissions ratio of the acceptor (535 nm) to donor (480 nm) molecule, conformational changes in MT can be inferred, including a relative decrease in FRET when MT is modified in such a way as to lose metal. Examples of this include exposure of the purified protein for several hours to the metal chelator EDTA (and NaCl) or the exposure of intact cells to medium saturated with gaseous NO.

Although these approaches permit quantitative assessment of the amount of cysteine modification, they give no information regarding the chemical nature or identity of the resulting products. Generally speaking, a fraction of these modified cysteines may harbor covalently adducted NO to form S-nitrosothiols (MT-SNO). In addition, other oxidation species may be formed such as disulfides or other higher S-oxides (sulfenic, sulfinic, and sulfonic acids).27'28 The latter pathway may be prevalent in highly oxygenated tissues such as lung. Direct analysis of S-NO within MT can be accomplished by a variety of techniques, one of which is described elsewhere in this series.29 This technique is based on UV-induced

25 A. Miyawaki, J. Llopos, R. Heim, J. M. McCaffrey, J. A. Adams, M. Ikura, and R. Y. Tsien, Nature

26 L. L. Pearce, R. E. Gandley, W. Han, K. Wasserloos, M. Stitt, A. J. Kanai, M. K. McLaughlin, B. R.

Pitt, and E. S. Levitan, Proc. Natl. Acad. Sci. U.S.A. 97, 477 (2000).

27 C. T. Aravindakumar, J. Ceulemans, and M. De Ley, Biochem. J. 344, 253 (1999).

28 A. R. Quesada, R. W. Byrnes, S. O. Krezoski, and D. H. Petering, Arch. Biochem. Biophys. 334,241

29 V. A. Tyurin, Y. Y. Tyurina, S.-X. Liu, H. Bayir, C. A. Hubel, and V. E. Kagan, Methods Enzymol.

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